Polysaccharide and uses thereof
AssigneeGlaxosmithkline Biologicals SA
InventorsMichael T. Kowarik, Michael L. Wetter, Stefan J. Kemmler, Micha A. Häuptle, Veronica Gambillara, Manuela Mally
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Abstract
Provided herein is an E. coli O polysaccharide, O25B. Also provided herein are prokaryotic host cells containing enzymes (e.g., glycosyltransferases) used in O25B production. The host cells provided herein produce O25B bioconjugates, wherein said bioconjugates contain O25B linked to a carrier protein. Further provided herein are compositions, e.g., pharmaceutical compositions, including O25B and/or bioconjugates containing O25B. Such compositions can be used as vaccines against infection with ExPEC, and may further include one or more additional bioconjugates.
Claims20
A recombinant prokaryotic host cell for producing an N-glycosylated carrier protein, comprising:
- (a) a nucleotide sequence encoding:
- (i) dTDP-Glucose 4,6-dehydratase;
- (ii) dTDP-6-Deoxy-D-glucose 3,5-epimerase;
- (iii) Glucose-1-phosphate thymidylyltransferase;
- (iv) dTDP-4-dehydrorhamnose 3,5-epimerase;
- (v) O antigen flippase;
- (vi) dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase;
- (vii) UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP α-1,6-glucosyltransferase;
- (viii) O antigen polymerase;
- (ix) O-acetyl transferase;
- (x) UDP-Glc:Rha-GlcNAc-UPP α-1,3-glucosyltransferase;
- and
- (xi) dTDP-Rha:
- GlcNAc-UPP α-1,3-rhamnosyltransferase;
- (b) a nucleotide sequence encoding an oligosaccharyl transferase;
- and
- (c) a nucleotide sequence encoding a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO:15).
A method of making an N-glycosylated carrier protein that comprises an E. coli O25B antigen, the method comprising:
- (a) culturing a recombinant prokaryotic host cell under conditions for producing the N-glycosylated carrier protein, wherein the prokaryotic host cell comprises:
- (i) a nucleotide sequence comprising a rfb gene cluster encoding the E. coli O25B antigen;
- (ii) a nucleotide sequence encoding an oligosaccharyl transferase;
- and
- (iii) a nucleotide sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X is any amino acid except Pro (SEQ ID NO:14);
- and
- (b) purifying the N-glycosylated carrier protein that comprises the E. coli O25B antigen.
Dependencies are inferred from the claim text.